Epidermal growth factor (EGF) is a naturally-occurring, relatively short, single-chain polypeptide, which was first isolated from the mouse submaxillary gland. A structurally very similar polypeptide was later detected and isolated from human urine at low (about 30 ng/ml) concentrations. Both mouse and human epidermal growth factors (the latter one also called urogastrone in some earlier publications) contain 53 amino acids. Thirty-seven of these are identical in the amino acid sequences of mouse epidermal growth factor (mEGF) and human epidermal growth factor (hEGF), as are the relative positions of the three disulfide bonds present in the structure. [Gregory, Nature, 257, 325 (1975); Gregory et al., Hoppe-Seyler's Z. Physiol. Chem., 356, 1765 (1975)]. The amino acid sequence of the 53 amino acid containing hEGF (.beta.-hEGF), as reported in the literature, is as follows: ##STR1##
The polypeptide also exists as a 52 amino acid form (gamma-hEGF) that lacks the C-terminal arginine residue found in .beta.hEGF.
The amino acid and nucleotide sequences of hEGF are, for example, disclosed in Hollenberg, "Epidermal Growth Factor-Urogastrone, A Polypeptide Acquiring Hormonal States"; eds., Academic Press, Inc., New York (1979), pp. 69-110; or Urdea et al., Proc. Natl. Acad. Sci. USA. 80, 7461 (1983).
A 48 amino acid containing form of hEGF (lacking C-terminal 5 amino acids) is described in the Japanese Patent Application 86146964, published 8 Feb. 1988 under No. 63003791.
The molecule in natural form contains disulfide linkages between residues 6-20, 14-31 and 33-42, and arises from an about 1200 amino acid precursor molecule consisting of eight EGF-like regions [see e.g. Bell et al., Nucleic Acid Research, 14, 21, 8427 (1986)]. A 48 amino acid containing form of rat EGF has recently been disclosed in the Japanese Patent Application 8736498, published 22 Aug. 1988, under No. 63202387. Both mEGF and hEGF, as well as their known analogs, exhibit similar pharmacological activities, although the extent or spectrum of activity may be different for different materials. In general EGF inhibits the secretion of gastric acid and promotes cell growth; therefore, it is targeted for therapeutic potential as an anti-ulcer agent and in external wound healing.
Since isolation from natural source is technically difficult, expensive, and time consuming, recent efforts have centered on the development of efficient recombinant methods for the production of EGF.
Of the hosts widely used for the production of heterologous proteins, probably E. coli and Saccharomyces cerevisiae (Baker's yeast) are the best understood. However, E. coli tends to produce EGF in its reduced form which is not stable in the presence of endogenous bacterial proteases. Attempts to overcome this problem, e.g. by employing a suitable leader sequence in order to produce an insoluble fusion protein which can be readily recovered from the cell paste resulted in other inconveniences, especially during purification of the product.
Yeasts can offer clear advantages over bacteria in the production of heterologous proteins, which include their ability to secrete heterologous proteins into the culture medium. Secretion of proteins from cells is generally superior to production of proteins in the cytoplasm. Secreted products are obtained in a higher degree of initial purity and their further purification is easier to contend with without cellular debris. In the case of sulfhydryl-rich proteins there is another compelling reason for the development of hosts capable of secreting them into the culture medium: their correct tertiary structure is produced and maintained via disulfide bonds. The secretory pathway of the cell and the extracellular medium are oxidizing environments which can support disulfide bond formation [Smith, et al., Science, 229, 1219 (1985)]. In contrast, the cytoplasm is a reducing environment in which disulfide bonds cannot form. Upon cell breakage, too rapid formation of disulfide linkages can result in random disulfide bond formation. Consequently, production of sulfhydryl rich proteins, such as EGF, containing appropriately formed disulfide bonds can be best achieved by transit through the secretory pathway.
Secretion of authentic biologically active human epidermal growth factor from S. cerevisiae is disclosed in European Patent Application Nos. 84104445.6 and 84303783.9, published Oct. 31, 1984 (No. 0 123 289) and Dec. 19, 1984 (No. 0 128 733), respectively. The cited patent applications contain no details as to the level of secretion or the purity of hEGF obtained. In an article published in Proc. Natl. Acad. Sci. USA, 81. 4642 (1984) Brake, inventor of the European Patent Application No. 84104445.6, and his co-workers give more details of their laboratory-scale experiments. hEGF is produced in S. cerevisiae by means of an expression cassette containing a DNA sequence encoding mature hEGF joined to sequences encoding the leader region ("pre-pro" segment) of the precursor of the yeast mating pheromone alpha-factor. In what appears to be the best experiment, hEGF was secreted into the shake flask culture medium in a concentration of about 4000 ng/ml. In view of the problems usually encountered with up-scaling the production of heterologous proteins in plasmid-based yeast systems, such as S. cerevisiae, there is no indication that hEGF production in S. cerevisiae could be at levels higher than those of that experimental system.
According to the prior art methods hEGF is produced and secreted from yeast in mature, usually 52 amino acid containing form.
To overcome the major problems associated with S. cerevisiae, e.g. loss of selection for plasmid maintenance and problems concerning plasmid distribution, copy number and stability in fermentors operated at high cell density, a yeast expression system based on the methylotrophic yeast Pichia pastoris has been developed. A key feature making this system unique lies with the promoter employed to drive heterologous gene expression. This promoter, which is derived from the methanol-regulated alcohol oxidase I (AOX1) gene of P. pastoris, is highly expressed and tightly regulated (see e.g. the European Patent Application No. 85113737.2, published June 4, 1986, under No. 0 183 071). Another key feature of the P. pastoris expression system is the stable integration of expression cassettes into the P. pastoris genome, thus significantly decreasing the chance of vector loss.
Although P. pastoris has been used successfully for the production of various heterologous proteins, e.g., hepatitis B surface antigen [Cregg et al., Bio/Technology 5, 479 (1987)], lysozyme and invertase [Digan et al., Developments in Industrial Microbiology 29, 59 (1988); Tschopp et al., Bio/Technology 5, 1305 (1987)], endeavors to produce other heterologous gene products in Pichia, especially by secretion, have given mixed results. At our present level of understanding of the P. pastoris expression system, it is unpredictable whether a given gene can be expressed to an appreciable level in this yeast or whether Pichia will tolerate the presence of the recombinant gene product in its cells. Further, it is especially difficult to foresee if a particular protein will be secreted by P. pastoris, and if it is, at what efficiency. Even for S. cerevisiae, which has been considerably more extensively studied than P. pastoris, the mechanism of protein secretion is not well defined and understood.